WO2011120474A1 - Metodo para inhibir la replicacion del vih en celulas de mamiferos y en humanos - Google Patents

Metodo para inhibir la replicacion del vih en celulas de mamiferos y en humanos Download PDF

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WO2011120474A1
WO2011120474A1 PCT/CU2011/000001 CU2011000001W WO2011120474A1 WO 2011120474 A1 WO2011120474 A1 WO 2011120474A1 CU 2011000001 W CU2011000001 W CU 2011000001W WO 2011120474 A1 WO2011120474 A1 WO 2011120474A1
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vimentin
keratin
agent
hiv
cells
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PCT/CU2011/000001
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English (en)
Spanish (es)
French (fr)
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WO2011120474A8 (es
WO2011120474A9 (es
Inventor
Celia Berta Fernandez Ortega
Anna Caridys RAMÍREZ SUÁREZ
Dionne Casillas Casanova
Taimi Emelia Paneque Guerrero
Raimundo UBIETA GÓMEZ
Marta DUBED ECHEVARRÍA
Leonor Margarita Navea Leyva
Lila Rosa Castellanos Serra
Carlos Antonio Duarte Cano
Viviana FALCÓN CAMA
Osvaldo Reyes Acosta
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Centro De Ingeniería Genética Y Biotecnología
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Priority to BR112012025092-2A priority Critical patent/BR112012025092B1/pt
Priority to EP11720370.3A priority patent/EP2554550B1/en
Priority to MX2012011424A priority patent/MX2012011424A/es
Priority to CN201180023063.3A priority patent/CN102884075B/zh
Priority to JP2013501620A priority patent/JP5925759B2/ja
Priority to AU2011235369A priority patent/AU2011235369B2/en
Priority to MYPI2012700711A priority patent/MY161923A/en
Application filed by Centro De Ingeniería Genética Y Biotecnología filed Critical Centro De Ingeniería Genética Y Biotecnología
Priority to US13/637,845 priority patent/US9205128B2/en
Priority to KR1020127028726A priority patent/KR101869917B1/ko
Priority to CA2794930A priority patent/CA2794930C/en
Priority to RU2012146542/10A priority patent/RU2593948C2/ru
Priority to ES11720370.3T priority patent/ES2604702T3/es
Publication of WO2011120474A1 publication Critical patent/WO2011120474A1/es
Publication of WO2011120474A9 publication Critical patent/WO2011120474A9/es
Priority to ZA2012/07761A priority patent/ZA201207761B/en
Publication of WO2011120474A8 publication Critical patent/WO2011120474A8/es
Priority to US14/943,089 priority patent/US20160129073A1/en
Priority to US14/943,096 priority patent/US20160106810A1/en
Priority to US14/943,106 priority patent/US10434137B2/en

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    • A61K31/713—Double-stranded nucleic acids or oligonucleotides
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    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
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    • A61K38/1709—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • A61K38/1748—Keratin; Cytokeratin
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    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
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    • C12N15/1131—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing against viruses
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Definitions

  • the present invention is related to the field of biomedicine, in particular with therapeutics against infections, and especially against infection by the human immunodeficiency virus (HIV). Describes a method to inhibit HIV replication by altering the cellular cytoskeleton, specifically proteins that are part of the intermediate filaments (Fl) of the cytoskeleton. It is also related to the use of agents that negatively modulate or alter the cellular cytoskeleton for the manufacture of drugs for the prevention and treatment of HIV infection.
  • HIV human immunodeficiency virus
  • multi-drug therapy does not completely eliminate HIV, and long-term treatment generally results in resistance to several drugs.
  • Half of the patients receiving combined anti-HIV therapy do not respond fully to treatment, mainly due to viral resistance to one or more drugs used.
  • Viral resistance has also been observed in newly infected individuals, which greatly limits the therapy options for these patients.
  • Adherence to treatment is one of the most controversial issues related to highly active antiretroviral therapy, and specifically with protease inhibitors, due to the rapidity with which resistance may occur if the drugs are taken irregularly or their administration is suspended.
  • the factors that condition non-adherence to treatment are several, ranging from intolerance to medications, discomfort of the regimens, therapeutic failure, interactions with medications, socioeconomic problems, among many others.
  • Combined therapy slows the progression to AIDS, however, it does not cure infected patients (Marsden MD, Zack JA 2009. J Antimicrob Chemoth 63: 7-10).
  • the present invention relates to the alteration of the cytoskeleton as a method to inhibit the replication and / or infection of HIV.
  • the cytoskeleton is a three-dimensional network that contributes to the integrity of the cell and performs different cellular functions. It consists of three types of structures: Microtubules, microfilaments and intermediate filaments (Fl). The latter are formed for a set of proteins, specific for each cell type, among which are vimentin and keratin-10.
  • Vimentin is a 58 kDa molecular weight protein (PM), which forms the so-called Fl and is typically expressed in endothelial cells of blood vessels, in some epithelial cells and in mesenchymal cells (Alberts B, Johnson A, Lewis J, Raff M, Roberts K, Walter P 2002. Molecular Biology of the Cell, 4th ed., Garland Publishing, New York). It is known that vimentin is a substrate of HIV protease (PR), and it has been proposed that the action of PR on this protein, and on other elements, can affect the structure of the cytoskeleton (Blanco R, Carrasco L, Windy I 2003. J Biol Chem 278: 1086-1093).
  • PR HIV protease
  • N-terminal peptides obtained by proteolytic cuts of vimentin, are capable of modifying the architecture of the cell nucleus, a phenomenon that is seen in HIV-infected cells (Shoeman RL, Wegtermann C, Hartig R , Traub P 2001. Mol Biol Cell 12: 143-154). Previous evidence suggests that HIV requires cleavage of the vimentin protein during its life cycle.
  • the present invention describes a method for inhibiting the replication and / or infection of HIV in mammalian cells by modulating or altering the structure of the cytoskeletal Fls in said mammalian cells.
  • the present invention is also directed to an agent that alters the cytoskeletal Fl to prevent or treat HIV infection.
  • the present invention provides a method to inhibit the replication of HIV in mammalian cells, said method consists in negatively altering or modulating the structure of the Fl of the cytoskeleton in mammalian cells.
  • said mammalian cells are the target cells of HIV infection.
  • the Fl comprise the vimentin and / or keratin-10 proteins.
  • the method comprises decreasing the amount of vimentin and / or keratin-10 of the Fl to negatively alter or modulate the Fl of the cytoskeleton (structure) and / or decrease the amount of vimentin and / or Keratin-10 free to form new Fl.
  • the method comprises the decrease (or inhibition) of the expression of the genes encoding the vimentin and / or keratin-10 to negatively alter or modulate the Fl of the cytoskeleton.
  • the alteration of the Fls is achieved by administering an effective therapeutic dose of an agent selected from a group consisting of polypeptides, peptides, nucleic acids and chemical compounds to said mammalian cells.
  • said agent is a peptide selected from the group of peptides identified as SEQ ID No. 1 to SEQ ID No. 10 and its counterparts.
  • said agent is an interfering RNA or an antisense oligonucleotide directed to the vimentin and / or keratin-10 genes or their transcripts.
  • said agent is a chemical compound or a lipid derivative.
  • the present invention provides an agent that negatively alters or modulates the cytoskeleton IFs to prevent or treat HIV infection.
  • said IFs comprise vimentin and / or keratin-10.
  • said agent induces a decrease in the amount of vimentin and / or keratin-10 in said IFs. In another preferred embodiment of such an agent, the agent decreases the expression of the genes encoding vimentin and / or keratin-10.
  • said agent is selected from a group consisting of polypeptides, peptides, nucleic acids and chemical compounds.
  • said agent comprises a peptide selected from the group of peptides identified as SEQ ID No. 1 to SEQ ID No. 10 and its homologs.
  • said agent is an interfering RNA or an antisense oligonucleotide, directed to the vimentin and / or keratin-10 genes or their transcripts.
  • said agent is an interfering RNA selected from a group consisting of a siRNA, shRNA and miRNA.
  • said interfering RNA consists of a sequence of 15 to 50 nucleotides complementary to a region of a messenger RNA of the vimentin and / or keratin-10 proteins, preferably 18 to 25 nucleotides.
  • said agent is a chemical compound and said compound is a lipid compound or a lipid derivative.
  • said lipid compound is prostaglandin cyclopentane 15 deoxy-A- 12 '1 -PGJ2 (15d-PGJ2).
  • the present invention provides a pharmaceutical composition for the prevention or treatment of HIV infection comprising the agent that negatively alters or modulates the IF of the cytoskeleton in accordance with the present invention as described above, and an excipient. pharmaceutically suitable.
  • said agent is selected from a group consisting of polypeptides, peptides, nucleic acids and chemical compounds that alter the IFs formed by vimentin and / or keratin-10.
  • said agent is a peptide selected from the group consisting of the peptides identified as SEQ ID No. 1 to SEQ ID No. 10 and their counterparts.
  • said agent is an interfering RNA or an antisense oligonucleotide, directed to the vimentin and / or keratin-10 genes or their transcripts.
  • said agent is for use in the treatment or prevention of HIV infection, or for use in the manufacture of a medicament for the treatment or prevention of HIV infection
  • Treatment or prevention of HIV infection includes inhibition or blocking of viral replication.
  • the interfering RNA is selected from a group consisting of siRNA, shRNA or miRNA.
  • the chemical compound is a lipid compound or a lipid derivative.
  • said lipid compound is cyclopentane deoxy-prostaglandin 15 A- 12 4 -PGJ2.
  • the present invention provides a pharmaceutical combination comprising an agent that alters the Fls of the cytoskeleton in accordance with that described in the present invention and at least one anti-HIV drug.
  • the protease inhibitor selected from the group consisting of: atazanavir (Reyataz TM), amprenavir (Agenerase TM
  • NRTIs reverse transcriptase inhibitors
  • efavirenz Stocrin TM
  • nevirapine nevirapine
  • etravirine Intelence TM
  • rilpivirine TMC-278
  • loviride R89439
  • Delavirdine Rescriptor TM
  • nucleoside reverse transcriptase inhibitors or nucleoside analogue reverse transcriptase inhibitors (lANTRs) such as: lamivudine (3TC or Epivir TM), abacavir (Ziagen TM) , zidovudine (AZT or Retrovir AZT TM), stavudine (d4T or Zerit TM), zalcitabine (ddC or Hivid TM), didanosine (ddl or Videx TM), emtricitabine (FTC or Emtriva TM), tenofovir (Viread TM), apricitabine ( AVX754), stampidine, elvucitabine (L-Fd4C), racivir, amdoxovir, functional derivatives of these drugs and combinations thereof such as: emtricitabine + tenofovir (Truvada TM), zidovudine
  • the pharmaceutical combination of the present invention may comprise combinations of the different classes of antiretroviral drugs listed above, such as the combinations: efavirenz + zidovudine + lamivudine, efavirenz + tenofovir + emtricitabine, lopinavir with ritonavir pulse + zidovudine + lamivudine, and lopinavir with ritonavir + tenofovir + emtricitabine pulse.
  • the agents and medicaments are administered simultaneously, separately or sequentially, as part of a dosage regimen.
  • the present invention provides a method for treating or preventing HIV infection in an individual in need thereof, which comprises administering to said individual an effective therapeutic dose of a pharmaceutical composition according to claims 20-26 or a pharmaceutical combination according to claims 27-28.
  • Figure 1 Relative intensity of human vimentin and keratin-10 proteins identified by comparative proteomics.
  • Panel A shows the decrease in vimentin protein in cultures treated with an extract with anti-HIV activity.
  • Panel B shows the decrease in keratin-10 protein in cultures treated with the extract with anti-HIV activity. Error bars mean standard deviation.
  • FIG. 1 Detection of vimentin and keratin-10 proteins in stably silenced cultures of each of these proteins.
  • the evaluation of vimentin (A) and keratin-10 (B) was performed by western blotting on the MT4 cell line, in MT4 vim (S ) and in MT4 K- io (s) - Silenced cultures showed decreased expression of proteins in relation to the culture of MT4.
  • the protein / 3-actin was used as a western blot normalizer. Each variant occupies two lanes.
  • K-10 is an abbreviation for keratin-10.
  • Figure 3 Inhibition of HIV-1 replication in MT4 v m (s) and MT4 K- io (s), as measured by p24 antigen.
  • Crops MT4 cells, MT4 v m (s) and MT4 K- io (S) were challenged with the Bru strain of HIV-1 at a multiplicity of infection (English multiplicity of infection, abbreviated moi) of 0.01.
  • a multiplicity of infection English multiplicity of infection, abbreviated moi
  • FIG. 4 Challenge test with the pLGW lentiviral vector in MT4, MT4 v m (s) and in MT4 K. io ( S ).
  • Cell cultures were transduced with a lentiviral vector that mimics the early stages of the HIV-1 replicative cycle after entry and carries the reporter gene that encodes GFP.
  • A MT4 cultures v m (s) and MT4 K- io (s) showed decreased percent fluorescent cells when transduced with the lentivirus, relative to cultures of MT4 cells. Error bars mean standard deviation.
  • B Histograms obtained from the analysis of the cultures by flow cytometry.
  • FIG. 7 Inhibition of HIV-1 replication, by peptides, in peripheral blood mononuclear cells (abbreviated Peripheral Blood Mononuclear Cells, abbreviated PBMC).
  • PBMCs peripheral blood mononuclear cells
  • Peptides inhibited HIV-1 replication in a dose-dependent manner.
  • Figure 8 Inhibition of HIV-2 replication by the peptide identified as SEQ ID No. 1.
  • PBMCs were pre-stimulated, treated with different concentrations of the peptides and infected with the CBL-20 strain of HIV-2.
  • the peptides inhibited HIV-2 replication in a dose-dependent manner.
  • Figure 9 Decrease of vimentin in the presence of the peptides identified as SEQ ID No. 1, 4, 5, 7, 8 and 9.
  • the MT4 cell line was incubated with the peptides, at 50 uM, for 24 hours.
  • the vimentin protein was detected using the western blot technique.
  • the vimentin bands appear diminished in peptide treated cultures.
  • Figure 10 Evaluation of the internalization of the peptides identified as SEQ ID No. 1 (A) and 3 (B) in the MT4 cell line. Bar graph representing the percentage of fluorescent cells indicative of peptide penetration at concentrations of 5, 10, 20 and 40 uM and different times in the MT4 cell line. Ce: untreated cells. Error bars mean standard deviation.
  • FIG. 11 Inhibition of HIV-1 replication by a lipid derivative. MT4 cells were incubated with different concentrations of cyclopentane prostaglandin 15 deoxy-A- 12,14 -PGJ2 (15d-PGJ2), and subsequently challenged with HIV-1 (Bru strain) at a moi of 0.01. Prostaglandin 15d-PGJ2 inhibited the replication of HIV-1. Error bars represent the standard deviation. Detailed description of the invention
  • the present invention solves the aforementioned problem, by describing a method for inhibiting HIV replication by negative modulation or alteration of the cellular cytoskeleton, specifically proteins that are part of the cytoskeleton Fl.
  • said Fl may be formed by acidic and basic keratins; vimentin; demines; glial fibrillary acid factor; peripheral; neurofilament protein (NF); internexine; filensin; phakinin; and laminins.
  • said Fl are formed by the vimentin and / or keratin proteins. More particularly, the Fl may be formed by vimentin and / or keratin-10. The intervention on the structure of these proteins causes an inhibition of virus replication in human cells.
  • the cytoskeleton as described herein refers to the scaffold or cellular skeleton contained in the cytoplasm and which is made of proteins.
  • the cytoskeleton is present in all cells; It plays an important role in cell division and intracellular transport. It consists of three main structures: microtubules, microfilaments and Fl.
  • the cytoskeleton provides the structure and shape to the cell. The elements of the cytoskeleton interact extensively and intimately with cell membranes.
  • Fls as defined herein are a family of related proteins that share common, structural and sequence characteristics.
  • the Fl have an average diameter of 10 nanometers, which is between actin (microfilaments) and microtubules.
  • Most types of Fl are cytoplasmic, but one type, lamines, is nuclear.
  • vimentin in this document refers to the member of the Fl protein family identified by the NCBI reference sequence: NP_003371.2, identified as SEQ ID No. 1.
  • Vimentin forms filamentous polymers in a series of assembly steps that begin with double antiparallel dimeros (or tetramers) to form filaments of unit length (in English, ULF) that are assembled longitudinally to form the entire filament.
  • Keratin refers to members of the family of fibrous structural proteins of Fl. Keratins form filamentous polymers in a series of assembly steps that begins with dimerization; the dimeros form tetramers and octamers and, eventually, ULF capable of joining at the ends to form the long filaments.
  • Each type I keratin is coexpressed with a specific type II keratin and each pair of keratin that is formed is characteristic and indicative of the differentiation and specialization of a particular type of epithelial cell.
  • keratin-10 refers to keratin-10 type I of the cytoskeleton, is the member of the Fl protein family identified in Swiss-Prot with the number: Q6EIZ0.1, with the sequence given in SEQ ID No. 12.
  • the term "gene,” herein refers to a DNA sequence, including but not limited to a DNA sequence that can be transcribed into mRNA that translates into polypeptide chains; transcribed in rRNA or tRNA or serve as a recognition site for enzymes and other proteins involved in DNA replication, transcription and regulation.
  • the term refers to any DNA sequence comprising several functionally linked DNA fragments such as a promoter region, a 5 'untranslated region (5' UTR), a coding region (which may or may not encode a protein) and a 3 'untranslated region (from English 3' UTR) comprising a polyadenylation site.
  • the 5'UTR, the coding region and the 3'UTR are transcribed into an RNA which, in the case of a gene encoding a protein, is translated into a protein.
  • the gene generally comprises introns and exons.
  • vimentin gene in this document refers to the gene encoding the vimentin protein or a homologue.
  • keratin-10 gene in this document refers to the gene that encodes the keratin-10 protein or a homologue.
  • alter used here to refer to the alteration of the Fl, as indicated herein refers to interference with the function or structural organization.
  • disruption or alteration may involve structural breakage, polymerization inhibition, formation and biosynthesis inhibition, including inhibition of the formation of primary, secondary and tertiary structures, etc. of proteins.
  • negative modulate which is used herein to refer to the negative modulation of the Fl, refers to changing or altering the structural function or organization so that it results in the loss or decrease of the biological function of said filaments.
  • intermediate cytoskeleton filaments in this document refers to intermediate filaments as a type of cytoskeleton element, of intermediate size compared to microfilaments and microtubules. These three elements contribute to structural integrity, cell shape and cell motility and organelles.
  • Type I and II acidic and basic keratins
  • Keratins also have subtypes that are unique to different epithelial cells
  • Type III Vimentin present in fibroblasts, endothelial cells and leukocytes; demines in muscle cells; glial fibrillary acid factor in astrocytes and other types of glia and peripheral peripheral nerve fibers
  • Type IV Neurofilament (NF) H (heavy), M (medium) and L (low), internexin filensin and fakinin proteins
  • type V laminins.
  • intermediate cytoskeleton filament structure in this document refers to the helical organization of tetramers of the filaments.
  • Each intermediate filament monomer consists of an alpha helical rod domain that connects the amino (head) and carboxyl (tail) terminals. The rod is wound around another filament to form a dimer. Terminals N and C of each filament are aligned. Some Fl form homodimers; other heterodimers. The dimers then form stepped tetramers that line head-tail. This tetramer is considered the basic subunit of the intermediate filament.
  • the final intermediate filament is a helical matrix of these tetramers.
  • treatment and “treat” refer to any use to remedy a condition or disease or its symptoms, or prevent the establishment of a condition or disease or symptoms, or prevent, hinder or reverse the progression of a condition or disease or its undesirable symptoms.
  • the term "effective therapeutic dose” herein refers to a non-toxic amount of the therapeutic agent sufficient to provide the desired therapeutic effect, for example, to treat, mitigate or prevent a desired disease or condition, or exhibit a therapeutic effect or Detectable preventive.
  • the effect can be detected by, for example, chemical markers or antigen levels.
  • the therapeutic effect also includes the reduction in physical symptoms.
  • the precise and effective amount for an individual will depend on the weight of the same and on their state of health, on the nature and extent of the condition and on the therapeutic or therapeutic combination selected for administration. Therefore, it is not useful to specify an exact amount effective in advance. However, the effective amount for a given situation can be determined by routine experimentation and is within the criteria of the clinician.
  • an effective dose will be about 0.01 mg / kg to 50 mg / kg or 0.05 mg / kg to 10 mg / kg of the polynucleotide or polypeptide compositions in the person to whom Is administered.
  • pharmaceutically acceptable carrier or excipient refers to an excipient for the administration of a therapeutic agent, such as a polypeptide, polynucleotide and other therapeutic agents.
  • a therapeutic agent such as a polypeptide, polynucleotide and other therapeutic agents.
  • the term refers to any pharmaceutical excipient that does not induce the production of antibodies harmful to the person receiving the composition, and which can be administered without excessive toxicity.
  • Suitable carriers can be large macromolecules that are slowly metabolized as proteins, polysaccharides, polylactic acids, polyglycolic acids, amino acid polymers, amino acid copolymers and inactive virus particles. Such vehicles are well known to be of ordinary skill in the art.
  • Pharmaceutically acceptable carriers of the therapeutic compositions may contain liquids such as water, saline, glycerol and ethanol.
  • auxiliary substances such as wetting or emulsifying agents, buffer substances and the like may be present in said vehicles.
  • auxiliary substances such as wetting or emulsifying agents, buffer substances and the like may be present in said vehicles.
  • auxiliary substances such as wetting or emulsifying agents, buffer substances and the like may be present in said vehicles.
  • homologue in this document, and when referring to a peptide, refers to a peptide having an amino acid sequence that shares at least a 70% sequence identity established by sequence alignment with, for example, Blast , etc., preferably at least 75%, more preferably at least 85%, 90% or even 95%, more preferably at least 97%, with the sequences identified as SEQ ID No. 1 to SEQ ID No. 10 and the ability to alter, negatively modulate and / or modify the cytoskeleton, specifically the proteins that make up the Fl of the cytoskeleton and more precisely the vimentin and keratin-10 proteins.
  • Appropriate homologs are peptides with conservative amino acid substitutions, preferably substitutions of less than 10% of the amino acids, more preferably less than 5%, less than 3% and more preferably even with less than 1% of the substituted amino acids. Preferably less than 10 amino acids are substituted, more preferably less than 5 and more preferably even less than 2 amino acids are substituted.
  • a conservative substitution is one in which an amino acid is replaced by another very similar amino acid, whose substitution has little or no effect on the activity of the peptide.
  • a "conservative substitution” is the substitution of one amino acid for another amino acid that has the same electronic net charge and approximately the same size and shape.
  • amino acids with aliphatic substituted or aliphatic amino acid side chains are approximately the same size when the total number of carbon and heteroatoms in their side chains differs by no more than about four. They have approximately the same shape when the number of branches in their side chains differs by no more than one. Amino acids with substituted phenyl or phenyl groups in their side chains are considered to have the same size and shape. Five groups of amino acids are listed below.
  • substitution of an amino acid in a polypeptide with another amino acid of the same group results in a Conservative substitution: Group I: glycine, alanine, valine, leucine, isoleucine, serine, threonine, cysteine and unnatural amino acids with aliphatic C1 C4 or C1 C4 aliphatic hydroxyl substituted chains (straight or mono-branched chain). Group II: glutamic acid, aspartic acid and unnatural amino acids with C1 C4 aliphatic side chains substituted carboxylic acid (unbranched or branched at one point).
  • Group III lysine, ornithine, arginine and unnatural amino acids with substituted C1 C4 aliphatic side chains (unbranched or branched at one point).
  • Group IV glutamine, asparagine and unnatural amino acids with aliphatic side chains with C1 C4 substituted amide (unbranched or branched at one point).
  • Group V phenylalanine, phenylglycine, tyrosine and tryptophan.
  • % sequence identity is defined herein as the percentage of nucleotides in a nucleic acid sequence that is identical to nucleotides in a nucleic acid sequence of interest, after aligning the sequences and optionally introducing gap, if it is necessary, to achieve the maximum percentage of sequence identity.
  • Computer methods and programs for alignments are well known in the state of the art.
  • nucleic acid sequence and “nucleotides” herein also comprise unnatural molecules based and / or derived from nucleic acid sequences, such as artificially modified nucleic acid sequences, nucleic acid-peptide, as well as nucleic acid sequences, comprising at least one modified nucleotide and / or unnatural nucleotide, such as inosine.
  • RNA interference refers to the process where an interfering RNA (iRNA) causes intracellular degradation of specific mRNA and can be used to interfere with the translation of a desired target mRNA transcript.
  • iRNA interfering RNA
  • interfering RNA refers to a double or single stranded RNA agent (iRNA), by which a small nucleic acid molecule used for RNA interference is understood.
  • iRNA RNA agent
  • Short iRNA agents that are approximately 15-30 nucleotides long are referred to as “small-interfering RNA” or siRNA.
  • Longer iRNA agents are generally referred to as “double stranded RNA” or dsRNA, other forms of iRNA agents are microRNA (miRNA) and short hairpin RNA molecules (shRNA).
  • the iRNA agents can be unmodified or chemically modified nucleic acid molecules.
  • the RNA agents can be chemically synthesized or expressed in a vector or enzymatically synthesized. The use of a chemically modified iRNA agent can improve one or more properties of an iRNA agent through increased degradation resistance, increased target or target specificity, improved cellular absorption and the like.
  • a DNA molecule that is transcribed to double stranded RNA or siRNA also provides RNA interference.
  • DNA molecules for transcribing double stranded RNA are described in U.S. Pat. Pat. No. 6,573,099 and in U.S. Patent Publications No. 20020160393 and 20030027783.
  • DNA molecules for transcribing siRNAs are reviewed in Tuschl and Borkhardt, Molecular Interventions, 2: 158 (2002).
  • antisense RNA in this document refers to any RNA that binds to mRNA with sufficient affinity to decrease the amount of protein translated from mRNA.
  • the amount of protein translated from the mRNA is preferably decreased by more than 20%; more preferably it decreased more than 50%, 70% and 80%; and more preferably decrease by more than 90%.
  • the methods and materials referred to antisense RNAs are well known in the state of the art.
  • expression herein refers to the transcription and stable accumulation of sense (mRNA) or antisense RNA derived from the nucleic acid fragment of the invention. Expression may also refer to the translation of mRNA into a polypeptide.
  • Antisense inhibition refers to the production of antisense RNA transcripts capable of suppressing the expression of the target protein.
  • expression inhibition is understood to mean silencing or regulating a gene or nucleic acid, which refers to a detectable decrease in transcription and / or translation of a target nucleic acid sequence, that is, the target sequence. of the iRNA, or a decrease in the amount or activity of the target sequence or protein compared to the normal level detected in the absence of the RNA or other nucleic acid sequence.
  • a detectable decrease can be as small as 5% or 10%, or as large as about 80%, 90% or 100%. More generally, a detectable decrease is approximately 20%, 30%, 40%, 50%, 60% or 70%.
  • lipid compound herein refers to analogous fatty acids derived from, for example, monounsaturated fatty acids, polyunsaturated fatty acids and lipids comprising triple bonds 1-6.
  • HIV is the human immunodeficiency virus, a retrovirus that causes immunodeficiency by attacking the body's CD4 + cells.
  • HIV in this document, includes any HIV, including all groups and subtypes (clades) of HIV-1 and HIV-2, for example groups M and O of HIV-1;
  • the invention encompasses each of the known subtypes; preferably of HIV-1.
  • replication in this document refers to the process in which a complementary chain of a nucleic acid molecule is synthesized by a polymerase enzyme.
  • replication is used herein in reference to a virus, it refers to the realization of a complete viral life cycle, where infectious viral particles or virions bind to the surface of the host cell (usually by binding to a specific cell surface molecule that gives the specificity of the infection). Once inside the cell, the virions are stripped and the viral genes begin to express the proteins necessary for genome replication and the synthesis of new proteins to make new capsids and nuclei that lead to the assembly of infectious viral particles of the progeny that They are capable of infecting and replicating in new host cells.
  • a viral life cycle is only completed if within a single cell infected by one or more viral particles or virions all stages occur for the production of the progeny of completely infectious viral particles
  • a cycle of complete viral life is that infectious viral particles containing the viral RNA enter a cell, the RNA is transcribed into DNA, the DNA is integrated into the chromosome of the host cell as a provirus and the infected cell produces the virion proteins and the virions are assembled carrying the complete viral genomic RNA in the infectious particles.
  • host cell refers to a cell that is used for the expression of a viral genome or for the propagation of a virus or a vector.
  • CD4 + cells refers to a general classification of T lymphocytes, referring to those that carry the CD4 antigen. Other CD4 + cells such as monocytes and macrophages are also included.
  • the present invention relates to methods that negatively modulate, modify or alter the IF of the cytoskeleton, preferably the Fl formed by the vimentin and / or keratin-10 proteins.
  • the method comprises decreasing the amount of vimentin and / or keratin-10 in the Fl.
  • the decrease of vimentin and / or keratin-10 can be affected in several ways.
  • a preferred embodiment is to decrease or inhibit the expression of the genes encoding vimentin and / or keratin-10.
  • the structure of the IF of the cytoskeleton can be altered by modifying the structure of the IF proteins, for example by rupture or changes in the tertiary structure, preferably the structure of vimentin and / or keratin-10.
  • the vimentin and keratin-10 cytoskeleton proteins were identified by a comparative proteomics study on MT4 cells treated with a fraction with anti-HIV activity, which was obtained from a human leukocyte extract.
  • a fraction with anti-HIV activity obtained from a human leukocyte extract.
  • the decrease in HIV infection is caused by the decrease and / or destabilization of vimentin and / or keratin-10 and / or the filaments containing these proteins.
  • an experimental system was used whose cellular route of entry is not mediated by the viral protein gp120.
  • Said system consists of a non-replicative lentiviral vector, based on HIV-1, which does not contain the gp120 protein and expresses the green fluorescent protein (abbreviated Green Fluorescent Protein, abbreviated GFP).
  • GFP green fluorescent protein
  • Fls are destabilized in MT4 silenced cells for vimentin (MT4 v (m)), as well as in silenced cells for keratin-10 (MT4) K- io (s)), producing an inhibition of "infection" by the HIV-1-based lentivirus (Example 3).
  • MT4 vm (S ) and MT4 K- io (s) cells were obtained by introducing specific RNA hairpins for the gene encoding each protein.
  • the alteration of the Fl can be achieved by an agent selected from a group consisting of polypeptides, peptides, nucleic acids and chemical compounds.
  • the agent is a peptide, more preferably the peptide is a peptide selected from the group identified as SEQ ID No. 1 to SEQ ID No. 10, and its homologues.
  • the agent is an interfering RNA or an antisense oligonucleotide directed to the vimentin and / or keratin-10 genes.
  • the agent is a chemical compound or a lipid derivative.
  • a suitable lipid compound is the prostaglandin 15d-PGJ2.
  • the present invention reveals methods for the treatment and / or prevention of HIV infection in human cells.
  • the methods involve the negative modulation of the vimentin and / or keratin-10 proteins in the cell in order to prevent or treat HIV infection in the cell.
  • Negative regulation occurs in an individual's HIV host cell, thus preventing or inhibiting effective infection of the individual's host cells.
  • the present invention also contemplates methods of treatment and / or prevention of HIV infection in a subject.
  • the inhibition of HIV infection by the method described in the present invention is applied at the cellular and whole organism level.
  • the term inhibition is used to refer to complete or partial inhibition of the infection.
  • the present invention describes the manipulation of the Fl, in particular the proteins of the cytoskeleton vimentin and keratin-10 to inhibit the replication of HIV, which offers the advantage over the current antiretroviral drugs that the possibilities of not producing or minimizing the possibilities of unleash viral resistance, since these proteins are not of viral origin but are cellular endogenous proteins.
  • the mechanisms of action of the drugs of the present invention act with a high capacity of inhibition by different routes to those described so far, so that the combination of these with the current therapeutic drugs specific for HIV infection could enhance the effectiveness of HIV treatments.
  • the use of the therapeutic agents of the present invention could be combined with novel therapeutic alternatives proposed in the state of the art, such as, for example, the transplantation of stem cells with modified endogenous genes.
  • the form of therapy of the present invention offers a new option to patients resistant to multiple drugs, which represent a high percentage among patients treated with the currently available therapy.
  • this invention surprisingly also manages to inhibit HIV infection without affecting cell viability, which adds even more novelty to the treatment of HIV infected patients.
  • the invention also relates to the use of agents that produce negative modulation or alteration of the cell cytoskeleton, specifically of the proteins that are part of the cytoskeleton Fl, more specifically of the vimentin and keratin-10 proteins for the production of a medicine for the prevention or treatment of HIV infection.
  • agents may be fused and / or conjugated to other molecules.
  • agents include peptide-like compounds, interfering RNAs and lipid compounds that produce the negative modulation or alteration of the cell cytoskeleton, of the Fl, and in particular of those that negatively modulate vimentin and / or keratin-10.
  • Negative modulation of vimentin and / or keratin-10 can be achieved through cell contact with an agent that negatively modulates vimentin or keratin-10.
  • the agent can be formulated to increase its ability to enter the cell if necessary. Negative modulation can be achieved by administering to an individual an agent that negatively modulates vimentin and / or keratin-10 in the subject's cells. Administration is performed so that the agent contacts the subject's cells, which are infected by HIV or could potentially become infected. Such cells are referred to herein as HIV host cells.
  • the administration of the agent entails contact with the host cell. Examples of such administration routes include parenteral routes and routes where the agent is administered through the subject's mucous membranes.
  • the cell is a CD4 + cell.
  • negative modulation or alteration can be achieved by directly affecting the vimentin and / or keratin-10, either by reduction of gene expression or protein synthesis, modification of the structure of the filaments that These proteins form, destabilization of this structure or by reduction of their activity / function.
  • negative modulation or alteration is understood as the inhibition of the level of the vimentin and / or keratin-10 protein in the cell, or the modification, destabilization, disassembly or even destruction of the structure of the cells. Fl that contain these proteins inside the cell.
  • Any known agent that negatively inhibits or modulates IFs and in particular vimentin and / or keratin-10 can be used to inhibit HIV infection, according to the method disclosed in the present invention.
  • HIV infection can also be inhibited by the use of peptide or polypeptide-type agents that negatively modulate or destabilize Fl, and in particular vimentin and / or keratin-10.
  • agents comprise endogenous proteins or proteins that are not normally found in the host cell. For example, mutated proteins, genetically engineered proteins, peptides, synthetic peptides, recombinant proteins, chimeric proteins, antibody fragments, humanized proteins, humanized antibodies, chimeric antibodies, modified proteins and fragments thereof.
  • the peptides are those identified in the Sequence Listing as SEQ ID No. 1 to SEQ ID No. 10.
  • the invention also contemplates the use of homologs of said peptides.
  • the peptides may be fused to another molecule, as for example, they may be fused to a penetrating peptide.
  • An agent useful for the prevention or treatment of HIV is one that inhibits the expression of the vimentin and / or keratin-10 gene, or protein synthesis, or filament structure containing vimentin or keratin-10.
  • a preferred agent according to the present invention is one that produces the silencing of the vimentin and / or keratin-10 gene or its transcripts with an iRNA, such as siRNA short interfering ribonucleic acid), shRNA (short hairpin ribonucleic acid) or miRNA (micro RNA).
  • RNA interference refers to a form of selective post-transcriptional gene silencing, which destroys the specific messenger RNA (mRNA), by a molecule that binds and inhibits mRNA processing. For example, you can inhibit mRNA translation or degrade it.
  • mRNA messenger RNA
  • RNA refers to any type of RNA, including but not limited to siRNA, shRNA, endogenous miRNA and artificial miRNA.
  • siRNA used herein refers to a nucleic acid that forms a double strand of RNA, which has the ability to reduce or inhibit the expression of the vimentin and / or keratin-10 gene.
  • the sequence of the siRNA may correspond to the complete sequence of the vimentin and / or keratin-10 gene.
  • the typical siRNA is at least 15 to 50 nucleotides long, preferably between 19 and 30 nucleotides.
  • a siRNA can be chemically synthesized, can be produced by in vitro transcription or can be produced within a cell specifically used for such production.
  • shRNA is used here as a type of siRNA. These shRNAs are composed of a short antisense chain, for example, between 19 and 25 nucleotides, followed by a 5 to 9 nucleotide loop and the analogous sense chain. Alternatively, the sense chain may precede the nucleotide loop and the antisense chain following it. ShRNAs function as RNAs and / or siRNA species, but differ in that shRNAs are fork-like structures to increase stability. These shRNAs, as well as other agents described herein, may be contained in plasmids, retroviruses and lentiviruses and expressed from, for example, the U6 polymerase III promoter or other promoter.
  • Delivery methods of the RNA-like agents to the white cell may include, for example, the injection of a composition containing the agent, or by contacting the cell directly, for example, a hematopoietic cell with a composition containing the RNA.
  • the RNA-like agent can be injected directly by any blood route such as a vein, artery, for example, by hydrodynamic injection or catheterization.
  • the RNA agent can be administered to specific organs or via systemic Colloidal dispersion systems can be used as delivery vehicles to increase the in vivo stability of the agents.
  • the agents can inhibit the expression of the vimentin and / or keratin-10 gene, such as an oligonucleotide or a nucleic acid analog.
  • these include, for example, a peptide-nucleic acid (PNA), a complementary pseudo-PNA (pc-PNA), "locked” nucleic acids (abbreviated locked nucleic acid, abbreviated LNA), and their derivatives.
  • Nucleic acid sequences encode proteins that act as transcriptional repressors, antisense molecules, ribozymes, small nucleic acid inhibitor sequences such as RNA, shRNA, siRNA, miRNA and antisense oligonucleotides.
  • the agent can take the form of any entity normally present or not at the levels that are being administered in the cell or organism. Agents such as chemicals, small molecules, aptamers, can be identified or generated to negatively modulate Fl and in particular vimentin and / or keratin-10.
  • aptamers are single chain nucleic acids, which have well-defined three-dimensional shapes allowing them to bind to the target molecules in a conceptually similar way to antibodies. Aptamers combine the optimal characteristics of small molecules and antibodies, including high specificity and affinity, chemical stability, low immunogenicity and the ability to attack protein-protein interactions.
  • the agent can function directly in the form in which it is administered, but it can also be modified, or used intracellularly to produce the negative modulation of vimentin and / or keratin-10.
  • the introduction of a nucleic acid sequence into the cell and its transcription results in the production of the nucleic acid and / or protein that inhibits Fl and in particular vimentin and / or keratin-10 within the cell.
  • the agent may comprise a vector.
  • the vectors can be episomal, for example, plasmids, vectors derived from viruses such as cytomegalovirus, adenovirus, etc., or they can be integrated into the genome of the white cell, for example, vectors derived from retroviruses, such as murine leukemia virus. Moloney, HIV-1, avian leukosis virus, etc. Vectors based on HIV or feline immunodeficiency virus can be used to transfect cells that are not dividing. Retrovirus combinations can be used. Many viral vectors or virus-associated vectors are known in the state of the art. Such vectors can be used as carriers of a nucleic acid construct to the cell.
  • the constructs can be integrated into non-replicative viral genomes similar to adenovirus (AAV), or herpes simplex, or others including retroviral and lentiviral vectors for infection or cell transduction.
  • AAV adenovirus
  • An HIV-based vector can be particularly useful in HIV host cells.
  • Another object of the invention is a pharmaceutical composition
  • the agents referred to in the present invention may be combined with each other or may be used in association with other therapeutic agents that may be, but are not limited to, known anti-HIV drugs, such as zidovudine (known as AZT). ).
  • the negative modulation of the Fl and in particular of the vimentin and / or the keratin-10 is applied in the present invention to cells that HIV is capable of infecting, with the purpose of preventing or reducing HIV infection. to that cell
  • the human cell is a CD4 + cell. The application of such negative modulation to an entire organism, human or primate, may constitute an effective therapeutic treatment for the organism against HIV infection.
  • Another embodiment of the present invention is a pharmaceutical combination comprising one an agent and a specific drug for the therapy of HIV infection.
  • the agents and drugs that are part of it can be administered simultaneously, separately or sequentially.
  • the present invention offers the advantage over current antiretroviral drugs that viral resistance does not occur or the chances of unleashing it are minimal, since the Fl and in particular the vimentin and keratin-10 proteins are not of viral origin, but which are cellular endogenous proteins.
  • the drugs of the present invention act with a high capacity of inhibition by different routes to those described so far in the state of the art, so that the combination of these with the current therapeutic drugs specific for HIV infection could enhance the effectiveness of HIV treatments.
  • the use of the therapeutic agents of the present invention could be combined with novel therapeutic alternatives proposed in the state of art as for example, the transplantation of stem cells with modified endogenous genes.
  • the present invention offers a new therapy to patients resistant to multiple drugs, which represent a high percentage among patients treated with the currently available therapy.
  • compositions of the invention once formulated, can be (1) administered directly to the subject; (2) administered ex vivo to cells derived from the subject; or (3) administered in vitro for the expression of recombinant proteins.
  • Direct administration of the compositions is generally performed by subcutaneous injection, intraperitoneally, intravenously or intramuscularly, or in the interstitial space of a tissue.
  • the compositions can also be administered in the nervous system.
  • Other modes of administration include topical, oral administration, suppositories and transdermal applications, needles and particle guns or pressure injector.
  • the treatment doses can be by single dose or multiple doses.
  • cells useful in ex vivo applications include, for example, stem cells, particularly hematopoietic, lymphoid cells, macrophages, dendritic cells or tumor cells.
  • nucleic acids for both ex vivo and in vitro applications can be accomplished by, for example, dextran-mediated transfection, calcium phosphate precipitation, polybrene-mediated transfection, electroporation, encapsulation of polynucleotide (s) in liposomes and by direct microinjection of DNA, well known in the state of the art.
  • Methods for introducing polynucleotides (such as siRNAs) into a cell are known in the state of the art.
  • Methods for the introduction of nucleic acid include for example transfection with calcium phosphate, DEAE-Dextran, electroporation or liposome-mediated transfection.
  • direct injection of the polynucleotides is employed.
  • a nucleic acid sequence is introduced into a cell by a vector, preferably a viral vector.
  • Said vector preferably comprises a retroviral, adenoviral, viral adeno-associated (AAV) or lentiviral vector.
  • Various methods are used to administer the therapeutic composition directly to a specific site in the body.
  • Specific administration mediated by receptors of therapeutic compositions containing an antisense polynucleotide, subgenomic polynucleotides or antibodies to specific tissues is also used.
  • the techniques of receptor-mediated DNA administration are described in, for example, Findeis et al., Trends in Biotechnol. (1993) 1: 202-205; Wu et al., J. Biol. Chem. (1994) 269: 542-46.
  • compositions containing polynucleotides are preferably administered in a range of about 100 ng to about 200 mg of the polynucleotides for local administration in a gene therapy protocol. Concentration ranges from about 500 ng to about 50 mg, about 1 pg to about 2 mg, about 5 pg to about 500 pg and about 20 pg to about 100 pg of polynucleotides can also be used during a gene therapy protocol . Factors such as the mode of action and the efficiency of transformation and expression are considerations that will affect the dose necessary for maximum effectiveness of the polynucleotides.
  • Example 1 Comparative proteomics of T4 cells treated with a leukocyte extract with anti-HIV activity.
  • the MT4 cell line was treated with a leukocyte extract with anti-HIV activity (Fernández-Ortega C; Dubed M; Ruibal I; Vilarrubia OL; Menéndez JC; Navea L ef al. 1996, Biotherapy 9: 33-40), and compared the protein expression profile against a control condition consisting of untreated cells.
  • the cells were lysed and centrifuged at 12,000 rpm by 20 min. The supernatant was separated and the sediment was subjected to a second lysis. It was centrifuged again at 12,000 rpm for 20 min.
  • the sample was slipped with ethyl alcohol and the sample was rented with polyacrylamide. Subsequently, deoxyribonucleic acid (DNA) was precipitated, and two-dimensional electrophoresis was carried out, which was performed on a gel with Tris-Tricine from 12.5 to 3% at 4 ° C.
  • DNA deoxyribonucleic acid
  • the images of the analytical gels were processed using the Melanie 5 program. From the gels prepared, the spots to be identified were cut and digested with trypsin. Proteolytic peptide extraction was performed for mass spectrometry analysis. The mass spectra were acquired on a hybrid mass spectrometer with orthogonal geometry QTOF-2 equipped with a "nanospray” ionization source.
  • ESI-MS / MS spectra were analyzed, and searches were performed for the identification of proteins in the non-redundant protein sequence base of the National Center for Biotechnology Information (abbreviated NCBI) of States United and at the base of the European Molecular Biology Laboratory (from the European Molecular Biology Laboratory, abbreviated EMBL) of Germany.
  • NCBI National Center for Biotechnology Information
  • EMBL European Molecular Biology Laboratory
  • a decrease in cytoskeleton proteins was observed, in particular of the proteins that form Fl (vimentin and keratin-10) ( Figure 1).
  • RNA interfering against vimentin and keratin-10 inhibits HIV infection.
  • the MT4 cell line was transduced using the pLenti-shRNA vim or pLenti-shRNA K- io lentiviral vectors carrying a sequence encoding an RNA hairpin that silences the vimentin protein and keratin-10 protein, respectively.
  • pLenti-shRNA vim or pLenti-shRNA K- io lentiviral vectors carrying a sequence encoding an RNA hairpin that silences the vimentin protein and keratin-10 protein, respectively.
  • four plasmids were packaged in the 293FT cell line, these are pLP1, pLP2, pLP / VSVG and the specific p-shRNA for vimentin or keratin-10.
  • PLP1 encodes the gene products of the gag / pol sequences of HIV-1.
  • PLP2 carries the HIV-1 REV protein gene sequence.
  • the pLPA / SVG encodes the surface protein of the vesicular stomatitis virus and the p-shRNA constitutes the genome of the lentiviral vector carrying the sequences encoding the specific RNA hairpins for vimentin (Ui-Tei K, Naito Y, Takahashi F, Haraguchi T et al., 2004 Nucleic Acids Research 32: 936-948) or for keratin-10 (Santa Cruz Biotechnology). All plasmids are amplified in the Escherichia coli XL-1 blue strain and selected in ampicillin. The four purified vectors per column, with transfection quality, are contacted with the 293FT packing cell line in the presence of polyethyleneimine.
  • the cells are incubated 48 hours at 37 ° C in a 5% CO2 atmosphere, and the virions are purified by ultracentrifugation at 20,000 x g.
  • MT4 cells were transduced and recombinant selection was performed by blasticidin resistance.
  • Recombinant clones were isolated by the limiting dilution method and cultured in 10% RPMI medium of fetal bovine serum (SFB) at 37 ° C under 5% C0 2 atmosphere and 95% relative humidity, until harvest.
  • S fetal bovine serum
  • Anti-HIV activity was evaluated using two challenge systems:
  • System A Stably silenced cells for vimentin protein (MT4 V im ( S )) or keratin-10 protein (MT4 K- io (s)) were grown in RPMI with 10% SFB at 37 ° C, in 5% C0 2 atmosphere and 95% relative humidity.
  • the challenge test with total virus was carried out in cultures of MT4 vim (S ), MT4 K- io (s) and MT4.
  • the Bru viral strain was used at a moi of 0.01, and the evaluation of replication was performed by determining the concentration of the p24 antigen in the culture supernatant, by the ELISA method.
  • MT4 and m (s) and MT4 K- 10 (S) cells showed a viral replication inhibition of about 90% with respect to MT4 cultures not silenced for this protein ( Figure 3).
  • MT4 cultures v m (s), MT4 K- io (s) and MT4 were challenged with a lentiviral vector containing part of the HIV-1 genome, without the genes associated with infectivity and the genes that code for the input (pLGW).
  • pLGW lentiviral vector containing part of the HIV-1 genome
  • four plasmids were packaged in the 293FT cell line, these are pLP1, pLP2, pLP / VSVG, and the pLGFP encoding the GFP protein.
  • PLP1 encodes the gene products of the gag / pol sequences of HIV-1.
  • PLP2 carries the HIV-1 REV protein gene sequence.
  • the pLP / VSVG encodes the surface protein of the vesicular stomatitis virus, and the pLGFP containing the packaging sequence, the RRE sequence (from the English Rev responsive element), and the LTRs regions (from the English long terminal repeats) deleted in the 3 ' end of HIV-1 constituting the genome of the lentiviral vector.
  • the expression of GFP was followed as an indicator of the completion of the stages of the replicative cycle after entry to integration. The results were followed by fluorescence microscopy, and a decrease of fluorescent cells was observed in cultures of MT4 v m (s) and MT4 K- io (s)> compared with MT4 cells (data not shown).
  • Example 3 Changes in the structure of the intermediate filaments of MT4 cells.
  • MT4 cells, MT4 v m (S) and MT4 K- io (s) were fixed in glutaraldehyde first 3.2%, for 1 hour at 4 ° C and postfixed in 2% osmium tetroxide for 1 hour at 4 ° C. Subsequently, they were washed with 0.1 M phosphate buffered saline (PBS) at pH 7.2 and dehydrated in increasing concentrations of ethanol (30, 50, 70 and 100%), for 10 minutes at a time, at 4 ° C. Inclusion was performed, ultrafine sections were prepared with an ultramicrotome (NOVA, LKB), with a thickness of 40-50 nm, and placed on 400-hole nickel gratings.
  • PBS phosphate buffered saline
  • Peptides were synthesized with amino acid sequences corresponding to human keratin-10 protein, human keratin 1 and human vimentin (Goldman RD, Khuon S, Hao Chou Y, Opal P, Steinert PM 1996, J Cell Biol 34: 971-983 ; Steinert PM, Yang JM, Bale SJ, Compton JG 1993, BBRC 197: 840-848).
  • One of the peptides has a penetrating peptide at its C-terminal (Vallespi MG, Fernandez JR, Torrens I, Garcia I, Garay H, Mendoza O et al. 2009. J Peptide Science 16: 40-47).
  • the anti-HIV activity of these peptides was evaluated using a challenge system with total virus, in the presence of different viral strains: HXB1 (HIV-1-III B) and Bru.
  • HXB1 HIV-1-III B
  • Bru The MT4 cell line was incubated with the peptide for 24 hours before the viral challenge. The tests were performed using moi values of 0.01 and 0.05, and nine replicates were conceived per experimental variant.
  • the value of the p24 viral antigen in cell cultures was determined by ELISA type assay, and the results were expressed in terms of percent viral inhibition or percent infection in relation to peptide concentration. An important inhibition of viral replication was observed in the presence of the peptides, both when the cultures were challenged with high viral concentration (SEQ ID No. 1, Figure 6A), and at a moi of 0.01 ( Figure 6B).
  • the IC50 of the peptides behaved at the nanomolar level.
  • Example 5 Inhibition of HIV-1 by peptides in peripheral blood mononuclear cells.
  • PBMCs were obtained from whole blood of healthy individuals by density gradient with Ficoll.
  • the cells were pre-stimulated for two days in RPMI medium with 20% SFB, 100 U / mL of interleukin 2 (IL-2) and 5 ug / mL of phytohemagglutinin (PHA). Subsequently, they were kept in PHA-free medium and 150,000 cells were seeded per well in. 96 well plates. After 24 hours, the peptides were added at different concentrations and the cultures were infected with the Bru strain at a moi of 0.01. The cultures were maintained for another 7 days, and every 3 days the medium was changed and the peptides were added again.
  • IL-2 interleukin 2
  • PHA phytohemagglutinin
  • PBMCs were obtained from whole blood of healthy individuals by density gradient with Ficoll.
  • the cells were pre-stimulated for two days in RPMI medium with 20% SFB, 100 U / mL of IL-2 and 5 ug / mL of PHA. Subsequently, they were kept in PHA-free medium and 150,000 cells were seeded per well in 96-well plates. After 24 hours, the peptides were added at different concentrations and the cultures were infected with the CBL-20 strain of HIV-2. The cultures were maintained for another 7 days, and every 3 days the medium was changed and the peptides were added again. The cultures were harvested and the supernatant was collected to assess the presence of the viral protein p24. The peptides inhibited dose-dependent HIV-2 replication (Figure 8).
  • Example 7 Decrease of vimentin in the presence of the peptides identified as SEQ ID No. 1, 4, 5, 7, 8 and 9.
  • the MT4 cell line was incubated with 50 ⁇ M of each peptide for 24 hours.
  • the vimentin protein was detected using the western blot technique.
  • the cell extracts resuspended in 1% sodium dodecyl sulfate (SDS) were applied on a 10% polyacrylamide gel, and subsequently transferred to a Hybond-P cellulose membrane.
  • SDS sodium dodecyl sulfate
  • mononoclonal antibodies anti-vimentin, and anti--- / 3 actin (as control) were used.
  • a mouse anti-lgG antibody conjugated to peroxidase was used as the secondary antibody.
  • the peroxidase enzyme activity was visualized by the use of diaminobenzidine in the presence of hydrogen peroxide and PBS.
  • the vimentin protein decreased in MT4 cells treated with the peptides ( Figure 9).
  • Example 8 Cellular penetration of the peptides identified as SEQ ID No. 1 and 3.
  • HeLaCD4 + cells were seeded in RPMI with 10% SFB, and incubated to ensure 60% confluence in the monolayer.
  • MT4 cells were seeded at 50,000 cells per well in RPMI with 10% SFB.
  • the peptides identified as SEQ ID No. 1 and 3 were reconstituted in water for injection and evaluated at concentrations of 5, 10, 20 and 40 ⁇ M.
  • the peptides were incubated for 24 hours at 37 ° C, under 5% CO 2 atmosphere, and penetration was evaluated at 15, 30 and 60 min. After each period, the cells were harvested and analyzed immediately on the flow cytometer. Three replicates were included per variant experimental. In Figure 10 it can be seen that the peptides penetrate the MT4 cell line alone.
  • Example 9 Lipid derivative that binds to vimentin inhibits HIV-1 replication.
  • Example 10 Effect of synthetic peptides and 15d-PGJ2 on PBMC of HIV-1 infected patients.
  • PBMCs were obtained from whole blood of HIV-1 infected individuals by density gradient with Ficoll.
  • the cells were pre-stimulated and treated with the peptides, in the same manner as in Example 5, or treated with 5 ⁇ M of 15d-PGJ2.
  • the replication evaluation was performed by determining the concentration of the p24 antigen in the culture supernatant, by the ELISA method.
  • P24 values decreased significantly in PBMCs treated with peptides or with the lipid derivative relative to untreated cells (Table 1), which is an indication of inhibition of HIV replication caused by treatment with these compounds .
  • Table 1 Percentage of inhibition of HIV-1 in PBMC from infected individuals treated ex vivo with the peptides or 15d-PGJ2
  • Peptide 1 (SEQ ID No. 1) 89.3
  • Peptide 6 (SEQ ID No. 6) 80.5
  • Peptide 7 (SEQ ID No. 7) 83.7 Peptide 8 (SEQ ID No. 8) 86.7
  • Peptide 9 (SEQ ID No. 9) 81.3
  • Peptide 10 (SEQ ID No. 10) 83.4
  • the structure of the Fl was analyzed by transmission electron microscopy, according to the methodology described in Example 3, and it was observed that the Fl are very shortened in the PBMC of the individuals who were treated with the peptides or with the derivative lipid, in relation to untreated cells.
  • Example 11 Interfering RNA against vimentin and keratin-10 inhibits HIV in PBMC of infected patients.
  • PBMCs were obtained from whole blood of HIV-1 infected individuals by density gradient with Ficoll.
  • the cells were pre-stimulated for two days in RPMI medium with 20% SFB, 100 U / mL of IL-2 and 5 ug / mL of PHA. Subsequently, they were maintained in a PHA-free medium and transduced with the lentiviral vectors pLenti-shRNAvim or pLenti-shRNA K- io, which carry a sequence encoding an RNA hairpin that silences the vimentin protein and keratin-10 protein, respectively.
  • Vectors were obtained as described in Example 2.
  • Replication evaluation was performed by determining the concentration of p24 antigen in the culture supernatant, by the ELISA method.
  • PBMCs silenced for the vimentin protein, or for the keratin-10 protein, showed a high inhibition of viral replication with respect to the non-silenced cultures for this protein (Table 2).
  • the structure of the Fl was analyzed by transmission electron microscopy, according to the methodology described in Example 3, and it was observed that the Fl were they find shortened in the PBMC of the individuals that were transduced with the lentiviral vectors, in relation to the untranslated cells.
  • Example 12 Treatment of HIV-1 infected patients with formulations containing the peptides identified as SEQ ID No. 1 and 2.
  • HIV-1 seropositive patients with less than one year of diagnosis and CD4 + T cell values greater than 350 cells / mm 3 , were treated with a formulation comprising the peptide identified as SEQ ID No. 1 or with a formulation that It comprises the one identified as SEQ ID No. 2.
  • the peptides were administered at 150 mg per day, and the patients were followed for 6 months in terms of viral load and CD4 + T cell count.
  • the viral load after treatment was undetectable in two of the patients, and decreased more than 1.5 log in six patients.
  • seven patients showed an increase in CD4 + T cells by more than 50 cells / mm 3 , while in one patient the value of CD4 + T cells decreased.
  • the structure of the PBMC Fl was analyzed by transmission electron microscopy, according to the methodology described in Example 3, in two of the patients treated with the peptide identified as SEQ ID No. 1 and in three of the treated patients with the peptide identified as SEQ ID No. 2. In all cases shortening was observed in Fl.

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